Method of inhibiting abcg2 and other treatment methods

ABSTRACT

Disclosed are methods of enhancing the chemotherapeutic treatment of tumor cells, reducing resistance of a cancer cell to a chemotherapeutic agent, a method of inhibiting ABCG2, Pgp, or MRP1 in a mammal afflicted with cancer, and a method of increasing the bioavailability of an ABCG2 substrate drug in a mammal. The methods comprise administering effective amounts of certain compounds to the mammal, for example, a compound of the formula (I): Formula (I), wherein R 1 , R 2 , R 3 , X 1 , X 2 , X 3 , a, and b are as described herein. Uses of these compounds in the preparation of a medicament are also disclosed. Also disclosed are compounds of formula (II), pharmaceutical compositions comprising such compounds and uses thereof.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/018,758, filed Jan. 3, 2008, the disclosure of whichis incorporated by reference.

BACKGROUND OF THE INVENTION

Multidrug resistance has long been recognized as a major obstacle tosuccessful cancer chemotherapy. The multidrug resistance transporterABCG2 (or Breast Cancer Resistance Protein 1, BCRP1), a member of theABC (ATP-binding cassette) family of membrane transport proteins, isbelieved to form a part of the maternal-fetal barrier, the blood-brainbarrier, and is known to limit oral absorption of some drugs (Robey etal., Cancer Metastasis Rev., 26: 39-57 (2007)). The normal physiologicfunctions of ABCG2 may be related to transport of a variety of naturalsubstances to prevent intracellular accumulation of toxic compounds.ABCG2 is also an important mediator of resistance to a variety ofanti-cancer drugs, including mitoxantrone, topotecan, irinotecan,flavopiridol, and methotrexate (Sarkadi et al., Physiol. Rev., 86:1179-1236 (2006); Krishnamurthy et al., Annu. Rev. Pharmacol. Toxicol.,46: 381-410 (2006); Szakacs et al., Nat. Rev. Drug Discov., 5: 219-34(2006); and Xu et al., Curr. Med. Chem., 14: 689-701 (2007)). Thus,inhibitors of ABCG2 activity could have important oncologic andpharmacologic applications.

Unfortunately, few, if any, clinically useful inhibitors of ABCG2activity have been reported. Thus, there exists a desire for compoundsthat can inhibit ABCG2 and in turn, increase the efficacy of adjuvantchemotherapy.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of enhancing the chemotherapeutictreatment of tumor cells in a mammal with a chemotherapeutic agent,which method comprises administering to the mammal an effective amountof the chemotherapeutic agent in conjunction with an effective amount ofa compound described herein that inhibits ABCG2 protein.

In an embodiment, the invention provides a method of reducing resistanceof a cancer cell to a chemotherapeutic agent by inhibiting ABCG2 in amammal, which method comprises administering to the mammal an effectiveamount of a compound described herein.

The invention further provides a method of inhibiting ABCG2, Pgp, and/orMRP1 in a mammal afflicted with cancer, which method comprisesadministering to the mammal an effective amount of a compound describedherein.

The invention also provides a method of increasing the bioavailabilityof an ABCG2 substrate drug in a mammal, which method comprisesadministering to the mammal an effective amount of the ABCG2 substratedrug in conjunction with an effective amount of a compound describedherein that inhibits ABCG2 protein.

The invention also provides uses of compounds described herein in thepreparation of a medicament for (i) enhancing the chemotherapeutictreatment of tumor cells in a mammal in combination with achemotherapeutic agent, (ii) inhibiting ABCG2 protein in a cancerpatient, (iii) reducing drug resistance of a chemotherapeutic agent in acancer patient, (iv) increasing the bioavailability of an ABCG2substrate drug in a cancer patient, (v) inhibiting MRP1 in a cancerpatient, or (vi) inhibiting Pgp in a cancer patient.

The invention also provides novel compounds of formula (II), describedbelow, pharmaceutical compositions comprising such compounds, and theirvarious uses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the formulas of certain botryllamides in accordance withan embodiment of the invention.

FIG. 2 depicts a reaction scheme to prepare compounds of formula (II) inaccordance with an embodiment of the invention: (1) is a condensationreaction; (2) acetylation with acetic anhydride and pyridine; (3)dehydration: DMSO, K₂CO₃, 98° C.; (4) NaOMe, MeOH, reflux or t-BuOK,THF, −40° C.; (5) octopamine HCl plus BICA-Na in 60% H₂SO₄.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, in accordance with an embodiment, a method ofenhancing the chemotherapeutic treatment of tumor cells in a mammal witha chemotherapeutic agent, which method comprises administering to themammal an effective amount of the chemotherapeutic agent in conjunctionwith an effective amount of a compound to inhibit ABCG2 protein, saidcompound being a compound of formula (I), to inhibit ABCG2 protein:

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl;

X¹ and X² are independently selected from the group consisting ofhydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxyC₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH), amido, formyl, andnitro; and

X³ and X⁴ are independently any of X¹ and X², cyano, or isonitrile; and

double bond “a” can be cis or trans and double bond “b” can be Z or E;

with the proviso that the compound is not botryllamide C, whereupon thechemotherapeutic treatment is enhanced.

By “enhancing the chemotherapeutic treatment” is meant that thechemotherapeutic agent has a greater effect (e.g., at least a 5%increase, at least a 10% increase, at least a 20% increase, at least a30% increase, at least a 40% increase, at least a 50% increase, at leasta 60% increase, at least a 70% increase, at least an 80% increase, etc.)in the presence of at least one compound described herein than in theabsence of that compound. Since ABCG2 is a mediator of resistance, if acompound described herein inhibits ABCG2, the cancerous cell is lessresistant to the chemotherapeutic agent, thereby making it moresusceptible to the cytotoxicity of the agent.

The present invention also provides a method of reducing resistance of acancer cell to a chemotherapeutic agent by inhibiting ABCG2 in a mammal,which method comprises administering to the mammal an effective amountof a compound of formula (I) to inhibit ABCG2 protein:

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl;

X¹ and X² are independently selected from the group consisting ofhydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxyC₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH), amido, formyl, andnitro; and

X³ and X⁴ are independently any of X¹ and X², cyano, or isonitrile; and

double bond “a” can be cis or trans and double bond “b” can be Z or E;

with the proviso that the compound is not botryllamide C, whereupon thedrug resistance is reduced.

By “reducing resistance of a chemotherapeutic agent” is meant thatcancer cells that are treated by the chemotherapeutic agent haveresistance reversed, development of resistance is reduced, or acombination thereof. For example, the resistance is reduced by at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, or at least 80%.

The chemotherapeutic agent described herein can be any cytotoxic drugthat is useful to kill cancer cells. In embodiments, the agent is anydrug in which there is resistance to a cancer cell upon administration.For example, the agent can be an antimetabolite (e.g., methotrexate), amitotic inhibitor (e.g., docetaxel, paclitaxel, vinblastine), analkylating agent (e.g., cisplatin), a cytotoxic antibiotic (e.g.,daunorubicin, doxorubicin, mitoxantrone), a topoisomerase inhibitor(e.g., topotecan, irinotecan), a tyrosine kinase inhibitor (e.g.,gefitinib), or any combination thereof. Specific examples of the ABCG2substrate drug include mitoxantrone, topotecan, irinotecan, SN-38,CPT-11, epirubin, flavopiridol, gefitinib, methotrexate, rhodamine,daunomycin, imatinib, axinitib, bosutinib, cediranib, dasatinib,dasatinib, erlotinib, lapatinib, lestaurtinib, nilotinib, semaxanib,vandetanib, vatalanib, doxorubicin, colchincine, vinblastine,paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, triplatintetranitrate, satraplatin, adriamycin, danofloxacin mesylate, docetaxel,and any combination thereof.

Any method known in the art can be used to measure the enhancement ofthe efficacy of the chemotherapeutic agent and/or the reduction ofresistance. The Examples section describes exemplary methods.Alternatively, cells can be contacted with a toxic chemotherapy drug,such as mitoxantrone or topotecan, in an amount that permits cellsurvival due to the resistance conferred by ABCG2. Cell viability can bemeasured by a colorimetric assay (Skehan et al., J. Natl. Cancer Inst.82: 1107 1112 (1990)), by counting cells with a cell counter, or byincorporation of tritiated thymidine.

The cells are then contacted with a compound of the invention thatinhibits ABCG2. The enhancement of the chemotherapeutic agent and/orreduction of resistance can then be detected by measuring the growthinhibition of cells, using a variety of means, such as IC₅₀measurements, vital staining, metabolite measurements, or confocalmicroscopy. Confocal microscopy can be used to determine whether aparticular drug has been retained or accumulated in the cell.

The invention further provides a method of inhibiting ABCG2 in a mammalafflicted with cancer, which method comprises administering to themammal an effective amount of a compound selected from the groupconsisting of

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl;

X¹ and X² are independently selected from the group consisting ofhydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxyC₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH), amido, formyl, andnitro; and

X³ and X⁴ are independently any of X¹ and X², cyano, or isonitrile; and

double bond “a” can be cis or trans and double bond “b” can be Z or E;

with the proviso that the compound is not botryllamide C,

whereupon ABCG2 is inhibited in the mammal.

Since ABCG2 has also been reported to be expressed at high levels in thedigestive tract and at the blood-brain barrier (Takano et al.,Pharmacol. Ther., 109: 137-61 (2006)), it is envisioned that ABCG2inhibitors can enhance bioavailability (e.g., oral bioavailability) andbrain penetration of ABCG2 substrate drugs, such as, e.g., topotecan.Thus, in an embodiment, the present invention provides a method ofincreasing the bioavailability of an ABCG2 substrate drug in a mammal,which method comprises administering to the mammal an effective amountof the ABCG2 substrate drug in conjunction with an effective amount of acompound to inhibit ABCG2 protein, said compound being selected from thegroup consisting of compound of formula (I) to inhibit ABCG2 protein:

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl;

X¹ and X² are independently selected from the group consisting ofhydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxyC₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH), amido, formyl, andnitro; and

X³ and X⁴ are independently any of X¹ and X², cyano, or isonitrile; and

double bond “a” can be cis or trans and double bond “b” can be Z or E;

with the proviso that the compound is not botryllamide C, whereupon thebioavailability of the ABCG2 substrate drug is improved.

Since CNS penetration can be enhanced by administration of a compound ofthe invention, such method is useful in the treatment of cancer, such asbrain tumors, CNS metastases, and/or gastrointestinal stromal tumors.

The ABCG2 substrate drug can be, for example, an antimetabolite (e.g.,methotrexate), a mitotic inhibitor (e.g., docetaxel, paclitaxel,vinblastine), an alkylating agent (e.g., cisplatin), a cytotoxicantibiotic (e.g., daunorubicin, doxorubicin, mitoxantrone), atopoisomerase inhibitor (e.g., topotecan, irinotecan), a tyrosine kinaseinhibitor (e.g., gefitinib), or any combination thereof. Specificexamples of the ABCG2 substrate drug include mitoxantrone, topotecan,irinotecan, SN-38, CPT-11, epirubin, flavopiridol, gefitinib,methotrexate, rhodamine, daunomycin, imatinib, axinitib, bosutinib,cediranib, dasatinib, dasatinib, erlotinib, lapatinib, lestaurtinib,nilotinib, semaxanib, vandetanib, vatalanib, doxorubicin, colchincine,vinblastine, paclitaxel, cisplatin, carboplatin, nedaplatin,oxaliplatin, iriplatin tetranitrate, satraplatin, adriamycin,danofloxacin mesylate, docetaxel, and any combination thereof. In someembodiments, the ABCG2 substrate drug is mitoxantrone, topotecan,irinotecan, flavopiridol, gefitinib, and/or methotrexate.

Pgp, MRP1, and ABCG2 are major contributors to multidrug resistance inmost cancer cells in culture (Szakacs et al., Nat. Rev. Drug Discov., 5:219-34 (2006)). ABCG2 has overlapping substrate specificity with MRP1and Pgp. Accordingly, in an embodiment, the present invention provides amethod of inhibiting MRP1 in a mammal afflicted with cancer, whichmethod comprises administering to the mammal an effective amount of acompound of formula (I) to inhibit ABCG2 protein:

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl;

X¹ and X² are independently selected from the group consisting ofhydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxyC₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH), amido, formyl, andnitro; and

X³ and X⁴ are independently any of X¹ and X², cyano, or isonitrile; and

double bond “a” can be cis or trans and double bond “b” can be Z or E;

with the proviso that the compound is not botryllamide C,

whereupon MRP1 is inhibited in the mammal.

All of the methods described herein have applicability to the treatmentof any type of cancer that over-expresses ABCG2 (and/or MRP1) and iscapable of being treated with a chemotherapeutic agent. Such cancersinclude, for example, leukemias (e.g., acute myeloid leukemia (AML),chronic myeloid leukemia (CML)), solid tumors (e.g., of the lung,endometrium, or digestive tract), melanomas, non-small cell lung cancertumors, colon tumors, prostate tumors, brain tumors, lymphomas, breasttumors, ovarian tumors, lung tumors, and stomach tumors.

For purposes of the present inventive methods, the mammal includes,without limitation, the order Rodentia, such as mice, and the orderLogomorpha, such as rabbits. It is preferred that the mammals are fromthe order Carnivora, including Felines (cats) and Canines (dogs). It ismore preferred that the mammals are from the order Artiodactyla,including Bovines (cows) and Swines (pigs) or of the orderPerssodactyla, including Equines (horses). It is most preferred that themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). An especially preferred mammalis the human.

In accordance with any of the embodiments of the invention, R¹ ishydrogen or C₁-C₆ alkyl, preferably methyl. In accordance with any ofthe embodiments of the invention, R² is hydrogen or C₁-C₆ alkyl,preferably methyl.

In accordance with any of the embodiments of the invention, X¹ and X²are independently selected from the group consisting of hydrogen andhalo. Any suitable halo (fluoro, chloro, bromo, or iodo) can beemployed, preferably bromo. For example, in embodiments of theinvention, one of X¹ and X² is hydrogen and the other of X¹ and X² isbromo.

In accordance with any of the embodiments of the invention, R³ ishydrogen or cyano. In accordance with any of the embodiments of theinvention, R² and R³, other than hydrogen, are in the ortho, meta, orpreferably para position of the phenyl rings to which they are attached.

In accordance with any of the embodiments of the invention, the compoundis selected from the group consisting of botryllamide A, botryllamide B,botryllamide D, botryllamide E, botryllamide F, botryllamide G,botryllamide I, botryllamide J, and any combination thereof,particularly botryllamide I or botryllamide J.

The present invention also provides an isolated or purified compoundselected from the group consisting of botryllamide I and botryllamide J.

The present invention further provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound selectedfrom the group consisting of botryllamide I and botryllamide J.

The present invention further provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and any of thecompounds described above.

The invention further provides a compound of formula (II):

wherein R² is selected from the group consisting of hydrogen, C₁-C₆alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, halo C₁-C₆ alkyl, andC₁-C₆ alkyl carbonyl;

X¹, X², and X⁶ are independently selected from the group consisting ofhydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxyC₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH), amido, formyl, andnitro; and

X³ and X⁴ are independently any of X¹ and X², cyano, or isonitrile;

X⁵ is selected from the group consisting of hydrogen, hydroxy, halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, haloC₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆ alkoxy carbonyl,C₁-C₆ alkylthio, mercapto (SH), amido, formyl, and nitro; and doublebond “a” can be cis or trans and double bond “b” can be Z or E; with theprovisos that (1) when X⁵ is hydrogen or hydroxy, X³ and X⁴ arehydrogen, X¹ and X² are hydrogen or halo, R² is hydrogen or methyl, X⁶is not alkoxy, e.g., not methoxy; and (2) when R², X¹, X², and X⁵ arehydrogen, X⁴ and X⁶ are hydroxy, then X³ is not NC.

In accordance with the invention, the alkyl group or alkyl portion of agroup containing an alkyl segment can be linear or branched, e.g.,methyl, ethyl, propyl, n-butyl, sec-butyl, t-butyl, n-pentyl,iso-pentyl, and the like. Alkenyl can be linear or branched. Aryl can bephenyl or naphthyl. Heteroaryl can be an aromatic group having aheteroatom such as O, N, or S.

In accordance with an embodiment of the invention, in the compound offormula (II), X¹, X², and X⁶ are independently selected from the groupconsisting of hydrogen, halo, C₁-C₆ alkyl, or C₁-C₆ alkoxy, particularlyhydrogen. In any of the embodiments of formula (II), X¹, X², and X⁶ arehalo. In any of the embodiments of formula (II), X¹, X², and X⁶ areC₁-C₆ alkyl. In any of the embodiments of formula (II), X¹, X², and X⁶are C₁-C₆ alkoxy.

In any of the embodiments of formula (II), X³ and X⁴ are independentlyany of X¹ and X², cyano, or isonitrile.

In any of the embodiments of formula (II), X³ and X⁴ are independentlyany of X¹ and X².

In any of the embodiments of formula (II), X⁵ is selected from the groupconsisting of hydrogen, hydroxy, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈cycloalkyl, hydroxy C₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂aryloxy, CNS, C₁-C₆ alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH),amido, formyl, and nitro, particularly, X⁵ is selected from the groupconsisting of hydrogen, hydroxy, halo, C₁-C₆ alkyl, and C₁-C₆ alkoxy. Inembodiments of formula (II), X⁵ is hydrogen. In embodiments of formula(II), X⁵ is hydroxy. In certain embodiments of formula (II), X⁵ is C₁-C₆alkyl. In certain other embodiments of formula (II), X⁵ is C₁-C₆ alkoxy.

The compounds of formula (II), which overlap with those of formula (I),are intended for the same uses and methods as described above for thecompounds of formula (I).

Accordingly, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound offormula (II). The invention also provides a method of enhancing thechemotherapeutic treatment of tumor cells in a mammal with achemotherapeutic agent, which method comprises administering to themammal an effective amount of the chemotherapeutic agent in conjunctionwith an effective amount of a compound of formula (II) to inhibit theABCG2 protein, a method of inhibiting ABCG2 in a mammal afflicted withcancer, which method comprises administering to the mammal an effectiveamount of a compound of formula (II), a method of reducing drugresistance to chemotherapeutic agents by inhibiting ABCG2 in a mammal,which method comprises administering to the mammal an effective amountof a compound of formula (II), a method of increasing thebioavailability of an ABCG2 substrate drug in a mammal, which methodcomprises administering to the mammal an effective amount of the ABCG2substrate drug in conjunction with an effective amount of a compound toinhibit ABCG2 protein, wherein said compound is a compound of formula(II), a method of inhibiting MRP1 in a mammal afflicted with cancer,which method comprises administering to the mammal an effective amountof a compound of formula (II), or a method of inhibiting Pgp in a mammalafflicted with cancer, which method comprises administering to themammal an effective amount of a compound of formula (II).

The invention also provides the use of a compound of formula (II) in thepreparation of a medicament for (i) enhancing the chemotherapeutictreatment of tumor cells in a mammal in combination with achemotherapeutic agent, (ii) inhibiting ABCG2 protein in a cancerpatient, (iii) reducing drug resistance of a chemotherapeutic agent in acancer patient, (iv) increasing the bioavailability of an ABCG2substrate drug in a cancer patient, (v) inhibiting MRP1 in a cancerpatient, or (vi) inhibiting Pgp in a cancer patient.

The present invention further provides a method of enhancing thechemotherapeutic treatment of tumor cells in a mammal with achemotherapeutic agent, which method comprises administering to themammal an effective amount of the chemotherapeutic agent in conjunctionwith an effective amount of a compound to inhibit ABCG2 protein, whereinsaid compound is selected from the group consisting of

and any combination thereof, whereupon the chemotherapeutic treatment isenhanced.

The present invention further provides a method of inhibiting ABCG2 in amammal, which method comprises administering to the mammal an effectiveamount of a compound selected from the group consisting of

and any combination thereof, whereupon ABCG2 is inhibited in the mammal.

The present invention also provides a method of reducing drug resistanceof cancer to chemotherapeutic agents by inhibiting ABCG2 in a mammal,which method comprises administering to the mammal an effective amountof a compound selected from the group consisting of

and any combination thereof, whereupon drug resistance of cancer isreduced in the mammal.

The present invention further provides a method of increasing thebioavailability of an ABCG2 substrate drug in a mammal, which methodcomprises administering to the mammal an effective amount of the ABCG2substrate drug in conjunction with an effective amount of a compound toinhibit ABCG2, wherein said compound is selected from the groupconsisting of:

and any combination thereof, whereupon the bioavailability is increased.

Generally, the compounds of the invention will be administered in apharmaceutical composition to an individual afflicted with a cancer.Those undergoing or about to undergo chemotherapy can be treated with atleast one compound described herein separately or in conjunction withother treatments, as appropriate. In therapeutic applications,compositions are administered to a patient in an amount sufficient toelicit an effective depression of ABCG2 activity thereby potentiatingthe cytotoxicity of the chemotherapeutic treatment. A dose adequate toaccomplish this is defined as an “effective amount,” which is also an“ABCG2 inhibiting effective amount.” Amounts effective for a therapeuticor prophylactic use will depend on, e.g., the stage and severity of thedisease being treated, the age, weight, and general state of health ofthe patient, and the judgment of the prescribing physician. The size ofthe dose will also be determined by the compound selected, method ofadministration, timing and frequency of administration as well as theexistence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular compound and the desiredphysiological effect. It will be appreciated by one of skill in the artthat various disease states may require prolonged treatment involvingmultiple administrations, perhaps using a series of different ABCG2inhibitors and/or chemotherapeutic agents in each or various rounds ofadministration.

Suitable chemotherapeutic agents administered in coordination with atleast one compound of the present invention include mitoxantrone,topotecan, irinotecan, flavopiridol, gefitinib, methotrexate, rhodamine,daunomycin, imatinib, doxorubicin, colchincine, vinblastine, paclitaxel,cisplatin, adriamycin, danofloxacin mesylate, and/or docetaxel. Thechemotherapeutic agent is administered in a dose sufficient to treat thecancer (e.g., cancer-treatment effective amount of a chemotherapeuticagent). Such doses are known in the art (see, for example, thePhysicians' Desk Reference (2004)). Such agents can be administeredusing techniques such as those described in, for example, Wasserman etal., Cancer, 36, pp. 1258-1268 (1975) and Physicians' Desk Reference,58th ed., Thomson PDR (2004).

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages that are lessthan the optimum dose of the compound of the present invention.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. The present inventivemethods can involve the administration of about 0.1 μg to about 50 mg ofat least one compound of the invention per kg body weight of theindividual. For a 70 kg patient, dosages of from about 10 μg to about200 mg of the compound of the invention would be more commonly used,depending on a patient's physiological response, e.g., as determined bymeasuring cancer-specific antigens or other measurable parametersrelated to the tumor load of a patient.

Any of the compounds of the invention can be administered in a dosesufficient to enhance the effect of the chemotherapeutic agent and/orreduce drug resistance in a cancer. A suitable dosage is that which willresult in a concentration of the compound of the invention in thecancerous cells to be treated sufficient to inhibit ABCG2 activity,e.g., from about 10 nM to 200 nM intracellularly, which can require anextracellular concentration of from about 10 μM to 50 μM. The dose canbe adjusted as necessary to enhance the effect of the chemotherapeuticagent and/or reduce drug resistance.

The pharmaceutical compositions for therapeutic treatment are intendedfor any suitable mode of administration, including parenteral, topical,oral, or local administration and generally comprise a pharmaceuticallyacceptable carrier and an amount of the active ingredient sufficient toreduce, and preferably prevent, the activity of ABCG2. The carrier canbe any of those conventionally used and is limited only bychemico-physical considerations, such as solubility and lack ofreactivity with the compound of the invention, and by the route ofadministration.

The pharmaceutically acceptable carrier (or excipient) is preferably onethat is chemically inert to the compound of the invention and one thathas no detrimental side effects or toxicity under the conditions of use.Such pharmaceutically acceptable carriers preferably include saline(e.g., 0.9% saline), Cremophor EL (which is a derivative of castor oiland ethylene oxide available from Sigma Chemical Co., St. Louis, Mo.)(e.g., 5% Cremophor EL/5% ethanol/90% saline, 10% Cremophor EL/90%saline, or 50% Cremophor EL/50% ethanol), propylene glycol (e.g., 40%propylene glycol/10% ethanol/50% water), polyethylene glycol (e.g., 40%PEG400/60% saline), and alcohol (e.g., 40% ethanol/60% water). Apreferred pharmaceutical carrier is polyethylene glycol, such as PEG400, and particularly a composition comprising 40% PEG 400 and 60% wateror saline. The choice of carrier will be determined in part by theparticular compound chosen, as well as by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention.

The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intraarterial, intramuscular, interperitoneal, rectal, andvaginal administration are merely exemplary and are in no way limiting.The pharmaceutical compositions can be administered parenterally, e.g.,intravenously, intraarterially, subcutaneously, intradermally,intrathecally, or intramuscularly. Thus, the invention providescompositions for parenteral administration that comprise a solution ofthe compound of the invention dissolved or suspended in an acceptablecarrier suitable for parenteral administration, including aqueous andnon-aqueous, isotonic sterile injection solutions.

Preferably a compound of the invention and a chemotherapeutic agent arecoadministered to the mammal. By “coadministering” is meantadministering the chemotherapeutic agent and a compound of the inventionsufficiently close in time such that a compound of the invention canenhance the effect of the chemotherapeutic agent. In this regard, acompound of the invention can be administered first and thechemotherapeutic agent can be administered second, or vice versa.Alternatively, a compound of the invention and the chemotherapeuticagent can be administered simultaneously. In addition, a combination ofcompounds of the invention can be administered, and one or more of thecompounds of the invention can be administered in combination withanother agent useful in the treatment of cancer.

Overall, the requirements for effective pharmaceutical carriers forparenteral compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986). Such compositions include solutions containing anti-oxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. The compounds of theinvention can be administered in a physiologically acceptable diluent ina pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol (for example intopical applications), or hexadecyl alcohol, glycols, such as propyleneglycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, suchas 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspolyethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils useful in parenteral formulations include petroleum, animal,vegetable, and synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral oil. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylene polypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-1′-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations typically will contain from about 0.5% orless to about 25% or more by weight of a compound of the invention insolution. Preservatives and buffers can be used. In order to minimize oreliminate irritation at the site of injection, such compositions cancontain one or more nonionic surfactants having a hydrophile-lipophilsbalance (HLB) of from about 12 to about 17. The quantity of surfactantin such formulations will typically range from about 5% to about 15% byweight. Suitable surfactants include polyethylene sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.

Topical formulations, including those that are useful for transdermaldrug release, are well known to those of skill in the art and aresuitable in the context of the present invention for application toskin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of a compound of the inventiondissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apre-determined amount of the compound of the invention, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations can include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant, suspending agent, or emulsifyingagent. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and cornstarch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising a compound of theinvention in an inert base, such as gelatin and glycerin, or sucrose andacacia, emulsions, gels, and the like containing, in addition to thecompound of the invention, such excipients as are known in the art.

A compound of the present invention, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. A compound of the invention is preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of the compounds of the invention can be about 0.01%to about 20% by weight, preferably about 1% to about 10% by weight. Thesurfactant must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such surfactants are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides canbe employed. The surfactant can constitute from about 0.1% to about 20%by weight of the composition, preferably from about 0.25% to about 5%.The balance of the composition is ordinarily propellant. A carrier canalso be included as desired, e.g., lecithin, for intranasal delivery.These aerosol formulations can be placed into acceptable pressurizedpropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also can be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations can be used to spray mucosa.

Additionally, a compound of the invention can be made into suppositoriesby mixing with a variety of bases, such as emulsifying bases orwater-soluble bases. Formulations suitable for vaginal administrationcan be presented as pessaries, tampons, creams, gels, pastes, foams, orspray formulas containing, in addition to the active ingredient, suchcarriers as are known in the art to be appropriate.

The concentration of a compound of the present invention in thepharmaceutical formulations can vary, e.g., from less than about 1%,usually at or at least about 10%, to as much as 20% to 50% or more byweight, and can be selected primarily by fluid volumes, and viscosities,in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 100mg of at least one compound of the invention. Actual methods forpreparing parenterally administrable compounds of the invention will beknown or apparent to those skilled in the art and are described in moredetail in, for example, Remington's Pharmaceutical Science (17th ed.,Mack Publishing Company, Easton, Pa., 1985).

It will be appreciated by one of ordinary skill in the art that, inaddition to the aforedescribed pharmaceutical compositions, a compoundof the invention can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes. Liposomes can serve totarget a compound of the invention to a particular tissue, such aslymphoid tissue or cancerous hepatic cells. Liposomes can also be usedto increase the half-life of a compound of the invention. Many methodsare available for preparing liposomes, as described in, for example,Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example illustrates a method of isolating botryllamides inaccordance with an embodiment of the invention. The marine ascidianBotryllus tyreus (75.5 g wet weight) is sequentially extracted with 1:1CH₂Cl₂-MeOH and 100% MeOH. Evaporation of the solvents under reducedpressure provides 4.8 g of crude extract. A 3 g aliquot of the extractis dissolved in 90% aq. MeOH and then extracted with n-hexane. Theaqueous MeOH fraction is further diluted with H₂O to provide a 60% aq.MeOH solution, which is then partitioned with CH₂Cl₂. The bioactiveCH₂Cl₂ fraction is separated by ODS (C₁₈) flash chromatography elutedwith a step gradient of 1:1 MeOH—H₂O, 4:1 MeOH—H₂O and 100% MeOH. Finalpurification by ODS HPLC elutes with a linear gradient from 20%CH₃CN-80% H₂O to 70% CH₃CN-30% H₂O over 50 minutes afforded botryllamideA (110 mg), botryllamide B (3.5 mg), botryllamide C (29 mg),botryllamide D (2.5 mg), botryllamide E (58 mg), botryllamide F (1.2mg), botryllamide G (9 mg), botryllamide H (4.0 mg), botryllamide I (4.7mg) and botryllamide J (1.6 mg).

Example 2

This example demonstrates a screening assay for ABCG2 inhibitors inaccordance with an embodiment of the invention.

Accumulation of pheophorbide a, a fluorescent ABCG2 substrate (Jonker etal., Proc. Natl. Acad. Sci. USA, 99: 15649-54 (2002) and Robey et al.,Cancer Res., 64: 1242-6 (2004)), formed the basis of the assay forinhibitors of ABCG2 activity (Henrich et al., J. Biomol. Screen, 11:176-83 (2006)). Briefly, NCI-H460/MX20 cells are transferred to blackwall, clear bottom 384-well polylysine-coated assay plates (Corning,Corning, N.Y.) and allowed to attach for several hours. Pheophorbide a(1 μM final concentration) is added immediately followed by compounds orvehicle (DMSO/PBS) control and incubated an additional 18 h. Afterremoval of medium and washing with PBS containing Ca²⁺ and Mg²⁺,fluorescence intensity is read on a Tecan Safire fluorescence platereader in bottom read mode, 395 nm excitation, and 670 nm emission. Eachplate has control wells containing 10 μM (final concentration) FTC. Dataare normalized to FTC and reported as % of FTC fluorescence.

Table 1 summarizes the activities of compounds in accordance with anembodiment of the invention in the pheophorbide a accumulation assay, aswell as other activities discussed in Example 3.

Example 3

This example demonstrates an assay to determine the ability of compoundsto sensitize cancer cells to killing by mitoxantrone in accordance withan embodiment of the invention.

The ability of compounds to sensitize NCI-H460/MX20 cells to killing bymitoxantrone is assessed as described in Henrich et al. (J. Biomol.Screen, 11: 176-83 (2006)). ABCG2 over-expressing cells or parentalcells are treated with mitoxantrone in the presence or absence of 10 μMcompound (or 1 μM FTC) and cell numbers assessed after 2 d by an XTTassay (Scudiero et al., Cancer Res., 48: 4827-33 (1988)). Final DMSOconcentration is 0.2% (v/v). Compounds in accordance with an embodimentof the invention can inhibit ABCG2-mediated transport usingBODIPY-prazosin as a substrate. For details of the test, see Robey etal., Br. J. Cancer, 89: 1971-8 (2003). This example also demonstratesthat exemplary compounds inhibit MRP1-mediated calcein efflux (Robey etal., 2003, vide supra and Alvarez et al., Mol. Pharmacol., 54: 802-14(1998)). Transfected HEK293 cells expressing ABCG2, Pgp, or MRP1 aretrypsinized and incubated in complete medium (phenol red-free Richter'smedium with 10% FCS and penicillin/streptomycin) containing 200 nMBODIPY-prazosin, 0.5 μg/ml rhodamine 123 or 200 nM calcein AM,respectively, in the presence or absence of the desired concentration ofinhibitor for 30 min at 37° C. The positive controls for inhibition ofABC transporters are 10 μM FTC for ABCG2, 3 μg/ml valspodar for Pgp and25 μM MK-571 for MRP1. Cells are then washed and incubated insubstrate-free medium continuing with or without inhibitor for 1 h.

TABLE 1 Effects of botryllamide compounds in multiple assays Pheophor-ABCG2 bide a ^(a)max MX Flow- Flow- Flow- activity, IC50 sensitizationBODIPY- ABCG2 Pgp, Fold MRP1, Fold Compound % (80 μM) (μM) (@ 10 μM)^(b)prazosin^(c) Flow-MX Increase^(d) Increase^(e) Botryllamide A 89.733 + + + 17.63 4.5 Botryllamide B  84.3 (40 μM) 11.2 + + + 1.95 3.26Botryllamide D 63.5 16.4 + + + 1.49 2.39 Botryllamide E 68.8 23.3 + + +0.76 1.96 Botryllamide F 81.0 16.7 + + + 0.59 3.33 Botryllamide G 123.7(40 μM) 6.9 + + + 0.68 2.1 Botryllamide I 71.3 41.4 + + + 0.8 3.14Botryllamide J 70.5 26.9 − + + 0.58 2.54 ^(a)% of FTC response ^(b)%NCI-H460/MX20 cell survival in the presence of compound and mitoxantrone^(c)BODIPY-prazosin efflux: treated/control ratio at 10 μM compound^(d)Pgp inhibition, rhodamine efflux: treated/control ratio at 10 μMcompound ^(e)MRP1 inhibition, calcein efflux: treated/control ratio at10 μM compound

Example 4

This example illustrates a method of isolation of naphthopyrones inaccordance with an embodiment of the invention. The compounds areisolated from two marine echinoderms, the sea star Capillastermultiradiatus and a taxonomically undefined crinoid. The marine samplesare sequentially extracted with 1:1 CH₂Cl₂-MeOH and 100% MeOH to provideorganic solvent extracts. A 1 g aliquot of the extract from Capillastermultiradiatus is subjected to a solvent-solvent partitioning scheme thatconcentrated the ABCG2 inhibitory activity into the EtOAc solublefraction. This fraction is chromatographed on Sephadex LH-20 with 1:1CH₂Cl₂-MeOH. Final purification by C₁₈ reversed-phase HPLC, eluting witha linear gradient from 75:25 MeOH/H₂O to 100% MeOH, provide compounds 1(1.0 mg), 3 (2.1 mg), 5 (2.4 mg) and 8 (3.1 mg).

A 2.0 g aliquot of the extract from the as yet undefined crinoid is alsosubjected to a solvent-solvent partitioning scheme that concentrated theactivity into the EtOAc and MeOtBu soluble fractions. The EtOAc fractionis chromatographed on Sephadex LH-20 with 2:5:1 hexane-CH₂Cl₂-MeOH andthe active fractions were subjected to C₁₈ reversed-phase HPLC as aboveto yield compounds 2 (1.8 mg), 4 (3.2 mg), 7 (5.6 mg), 9 (5.3 mg) and 10(2.1 mg). The MeOtBu fraction is chromatographed on Sephadex LH-20 with1:1 CH₂Cl₂-MeOH and further purified by HPLC as described above to yieldcompounds 6 (1.0 mg) and 11 (1.1 mg).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of enhancing the chemotherapeutic treatment of tumor cellsin a mammal with a chemotherapeutic agent, which method comprisesadministering to the mammal an effective amount of the chemotherapeuticagent in conjunction with an effective amount of a compound of formula(I) to inhibit ABCG2 protein:

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl; X¹ and X² areindependently selected from the group consisting of hydrogen, halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, haloC₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆ alkoxy carbonyl,C₁-C₆ alkylthio, mercapto (SH), amido, formyl, and nitro; and X³ and X⁴are independently any of X¹ and X², cyano, or isonitrile; and doublebond “a” can be cis or trans and double bond “b” can be Z or E; with theproviso that the compound is not botryllamide C, whereupon thechemotherapeutic treatment is enhanced.
 2. A method of inhibiting ABCG2in a mammal afflicted with cancer, which method comprises administeringto the mammal an effective amount of a compound of formula (I):

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl; X¹ and X² areindependently selected from the group consisting of hydrogen, halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, haloC₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆ alkoxy carbonyl,C₁-C₆ alkylthio, mercapto (SH), amido, formyl, and nitro; and X³ and X⁴are independently any of X¹ and X², cyano, or isonitrile; and doublebond “a” can be cis or trans and double bond “b” can be Z or E; with theproviso that the compound is not botryllamide C, whereupon ABCG2 isinhibited.
 3. A method of reducing drug resistance to chemotherapeuticagents by inhibiting ABCG2 in a mammal, which method comprisesadministering to the mammal an effective amount of a compound of formula(I):

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl; X¹ and X² areindependently selected from the group consisting of hydrogen, halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, haloC₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆ alkoxy carbonyl,C₁-C₆ alkylthio, mercapto (SH), amido, formyl, and nitro; and X³ and X⁴are independently any of X¹ and X², cyano, or isonitrile; and doublebond “a” can be cis or trans and double bond “b” can be Z or E; with theproviso that the compound is not botryllamide C, whereupon resistance ofthe chemotherapeutic agent is reduced in the mammal.
 4. The method ofclaim 1, wherein the chemotherapeutic agent is selected from the groupconsisting of an antimetabolite, a mitotic inhibitor, an alkylatingagent, a cytotoxic antibiotic, a topoisomerase inhibitor, a tyrosinekinase inhibitor, and any combination thereof.
 5. The method of claim 1,wherein the chemotherapeutic agent is selected from the group consistingof mitoxantrone, topotecan, irinotecan, flavopiridol, gefitinib,methotrexate, rhodamine, daunomycin, imatinib, doxorubicin, colchincine,vinblastine, paclitaxel, cisplatin, adriamycin, danofloxacin mesylate,docetaxel, and a combination thereof.
 6. A method of increasing thebioavailability of an ABCG2 substrate drug in a mammal, which methodcomprises administering to the mammal an effective amount of the ABCG2substrate drug in conjunction with an effective amount of a compound toinhibit ABCG2 protein, wherein said compound is of formula (I):

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl; X¹ and X² areindependently selected from the group consisting of hydrogen, halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, haloC₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆ alkoxy carbonyl,C₁-C₆ alkylthio, mercapto (SH), amido, formyl, and nitro; and X³ and X⁴are independently any of X¹ and X², cyano, or isonitrile; and doublebond “a” can be cis or trans and double bond “b” can be Z or E; with theproviso that the compound is not botryllamide C, whereupon thebioavailability is increased.
 7. A method of inhibiting MRP1 in a mammalafflicted with cancer, which method comprises administering to themammal an effective amount of a compound of formula (I):

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl; X¹ and X² areindependently selected from the group consisting of hydrogen, halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, haloC₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆ alkoxy carbonyl,C₁-C₆ alkylthio, mercapto (SH), amido, formyl, and nitro; and X³ and X⁴are independently any of X¹ and X², cyano, or isonitrile; and doublebond “a” can be cis or trans and double bond “b” can be Z or E; with theproviso that the compound is not botryllamide C, whereupon MRP1 isinhibited in the mammal.
 8. A method of inhibiting Pgp in a mammalafflicted with cancer, which method comprises administering to themammal an effective amount of a compound of formula (I):

wherein R¹, R², and R³ are independently selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆alkyl, halo C₁-C₆ alkyl, and C₁-C₆ alkyl carbonyl; X¹ and X² areindependently selected from the group consisting of hydrogen, halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, haloC₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy, CNS, C₁-C₆ alkoxy carbonyl,C₁-C₆ alkylthio, mercapto (SH), amido, formyl, and nitro; and X³ and X⁴are independently any of X¹ and X², cyano, or isonitrile; and doublebond “a” can be cis or trans and double bond “b” can be Z or E; with theproviso that the compound is not botryllamide C, whereupon Pgp isinhibited in the mammal.
 9. The method of claim 6, wherein the ABCG2substrate drug is selected from the group consisting of anantimetabolite, a mitotic inhibitor, an alkylating agent, a cytotoxicantibiotic, a topoisomerase inhibitor, a tyrosine kinase inhibitor, andany combination thereof.
 10. The method of claim 6, wherein the ABCG2substrate drug is selected from the group consisting of mitoxantrone,topotecan, irinotecan, SN-38, CPT-11, epirubin, flavopiridol, gefitinib,methotrexate, rhodamine, daunomycin, imatinib, axinitib, bosutinib,cediranib, dasatinib, dasatinib, erlotinib, lapatinib, lestaurtinib,nilotinib, semaxanib, vandetanib, vatalanib, doxorubicin, colchincine,vinblastine, paclitaxel, cisplatin, carboplatin, nedaplatin,oxaliplatin, triplatin tetranitrate, satraplatin, adriamycin,danofloxacin mesylate, docetaxel, and any combination thereof.
 11. Themethod of claim 1, wherein cancer is treated and the cancer is selectedfrom the group consisting of leukemia, a solid tumor, a melanoma, anon-small cell lung cancer tumor, a colon tumor, a prostate tumor, abrain tumor, a lymphoma, a breast tumor, an ovarian tumor, a lung tumor,and a stomach tumor.
 12. The method of claim 1, wherein R¹ is hydrogenor C₁-C₆ alkyl.
 13. The method of claim 12, wherein R¹ is C₁-C₆ alkyl.14. The method of claim 13, wherein R¹ is methyl. 15-25. (canceled) 26.The method of claim 1, wherein the compound is selected from the groupconsisting of botryllamide A, botryllamide B, botryllamide D,botryllamide E, botryllamide F, botryllamide G, botryllamide I,botryllamide J, and any combination thereof.
 27. (canceled)
 28. Anisolated or purified compound selected from the group consisting ofbotryllamide I and botryllamide J.
 29. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound selectedfrom the group consisting of botryllamide I and botryllamide J. 30.(canceled)
 31. A method of enhancing the chemotherapeutic treatment oftumor cells in a mammal with a chemotherapeutic agent, which methodcomprises administering to the mammal an effective amount of thechemotherapeutic agent in conjunction with an effective amount of acompound to inhibit ABCG2 protein, wherein said compound is selectedfrom the group consisting of

and any combination thereof, whereupon the chemotherapeutic treatment isenhanced.
 32. A method of inhibiting ABCG2 in a mammal, which methodcomprises administering to the mammal an effective amount of a compoundselected from the group consisting of

and any combination thereof, whereupon ABCG2 is inhibited in the mammal.33. A method of reducing drug resistance of cancer to chemotherapeuticagents by inhibiting ABCG2 in a mammal, which method comprisesadministering to the mammal an effective amount of a compound selectedfrom the group consisting of

and any combination thereof, whereupon development of drug resistance ofcancer is reduced in the mammal.
 34. A method of increasing thebioavailability of an ABCG2 substrate drug in a mammal, which methodcomprises administering to the mammal an effective amount of the ABCG2substrate drug in conjunction with an effective amount of a compound toinhibit ABCG2, wherein said compound is selected from the groupconsisting of:

and any combination thereof, whereupon the bioavailability is increased.35-37. (canceled)
 38. A compound of formula (II):

wherein R² is selected from the group consisting of hydrogen, C₁-C₆alkyl, C₃-C₈ cycloalkyl, hydroxy C₁-C₆ alkyl, halo C₁-C₆ alkyl, andC₁-C₆ alkyl carbonyl; X¹, X², and X⁶ are independently selected from thegroup consisting of hydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈cycloalkyl, hydroxy C₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂aryloxy, CNS, C₁-C₆ alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH),amido, formyl, and nitro; and X³ and X⁴ are independently any of X¹ andX², cyano, or isonitrile; X⁵ is selected from the group consisting ofhydrogen, hydroxy, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl,hydroxy C₁-C₆ alkyl, halo C₁-C₆ alkyl, C₁-C₆ alkyl carbonyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₆-C₁₁ aryl, C₃-C₇ heteroaryl, C₆-C₁₂ aryloxy,CNS, C₁-C₆ alkoxy carbonyl, C₁-C₆ alkylthio, mercapto (SH), amido,formyl, and nitro; and double bond “a” can be cis or trans and doublebond “b” can be Z or E; with the provisos that (1) when X⁵ is hydrogenor hydroxy, X³ and X⁴ are hydrogen, X¹ and X² are hydrogen or halo, R²is hydrogen or methyl, X⁶ is not alkoxy; and (2) when R², X¹, X², and X⁵are hydrogen, X⁴ and X⁶ are hydroxy, then X³ is not NC.
 39. The compoundof claim 38, wherein R² is hydrogen or C₁-C₆ alkyl.
 40. The compound ofclaim 38, wherein R² is C₁-C₆ alkyl. 41-53. (canceled)
 54. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim
 38. 55. A method of enhancing thechemotherapeutic treatment of tumor cells in a mammal with achemotherapeutic agent, which method comprises administering to themammal an effective amount of the chemotherapeutic agent in conjunctionwith an effective amount of a compound of claim 38 to inhibit the ABCG2protein.
 56. A method of inhibiting ABCG2 in a mammal afflicted withcancer, which method comprises administering to the mammal an effectiveamount of a compound of claim
 38. 57. A method of reducing drugresistance to chemotherapeutic agents by inhibiting ABCG2 in a mammal,which method comprises administering to the mammal an effective amountof a compound of claim
 38. 58. A method of increasing thebioavailability of an ABCG2 substrate drug in a mammal, which methodcomprises administering to the mammal an effective amount of the ABCG2substrate drug in conjunction with an effective amount of a compound toinhibit ABCG2 protein, wherein said compound is a compound of claim 38.59. A method of inhibiting MRP1 in a mammal afflicted with cancer, whichmethod comprises administering to the mammal an effective amount of acompound of claim
 38. 60. A method of inhibiting Pgp in a mammalafflicted with cancer, which method comprises administering to themammal an effective amount of a compound of claim
 38. 61. (canceled)